I. Field of the Invention
The present invention relates to the fields of cell biology, immunology and pathology. More specifically, the invention deals with optimized sequences of oligodeoxynucleotides (ODNs) that modulate immune responses, in particular, those involving Toll-like receptors (TLRs).
II. Related Art
Reaction to certain motifs in bacterial DNA is an important function of natural immunity. Bacterial DNA has long been known to be mitogenic for mammalian B lymphocytes (B cells), whereas mammalian DNA generally is not. The discovery that this immune recognition was directed to specific DNA sequences centered on a motif containing an unmethylated CpG dinucleotide opened the field to molecular immunologic approaches (Krieg et al., 1995). The immunostimulatory effects of so-called CpG DNA can be reproduced using synthetic oligodeoxynucleotides (ODN) containing CpG dinucleotides in the context of certain preferred flanking sequence, a CpG motif. CpG-containing ODN (CpG-ODN) have been reported to exert a number of effects on various types of cells of the immune system, including protecting primary B cells from apoptosis, promotion of cell cycle entry, and skewing an immune response toward a Th1-type immune response, e.g., induction of interleukin 6 (IL-6), interleukin 12 (IL-12), γ interferon (IFN-γ), activation of antigen-specific cytolytic T lymphocytes (CTL), and induction in the mouse of IgG2a.
It has been reported that the immunomodulatory effects of CpG DNA involve signaling by Toll-like receptor 9 (TLR9). It is believed that CpG DNA is internalized into a cell via a sequence-nonspecific pathway and traffics to the endosomal compartment, where it interacts with TLR9 in a sequence-specific manner. TLR9 signaling pathways lead to induction of a number of immune-function related genes, including notably NF-κB, among others.
The TLRs are a large family of receptors that recognize specific molecular structures that are present in pathogens (pathogen-associated molecular patterns or PAMPs) and are also termed pattern recognition receptors (PRRs). Immune cells expressing PRRs are activated upon recognition of PAMPs and trigger the generation of optimal adaptive immune responses. PRRs consisting of 10 different TLR subtypes, TLR1 to TLR10, have been described. Such TLRs have been described to be involved in the recognition of double-stranded RNA (TLR3), lipopolysaccharide (LPS) (TLR4), bacterial flagellin (TLR5), small anti-viral compounds (TLR7 and TLR8), and bacterial DNA or CpG ODN (TLR9). Reviewed in Uhlmann et al. (2003). In addition, RNA molecules were recently identified that are believed to interact with and signal through TLR7 and TLR8 (PCT/US03/10406). Such immunostimulatory RNA molecules are believed to have a base sequence that includes at least one guanine and at least one uracil. The immunostimulatory G,U-rich RNA does not require a CpG motif as described for TLR9. The corresponding class of RNA molecules found in nature is believed to be present in ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA).
Following the discovery of immunostimulatory CpG DNA, a number of reports appeared describing short DNA sequences with TLR-restricted immunoinhibitory effects. It has long been known that poly-G sequences were immunoinhibitory. PCT application WO 00/14217 describes ODN containing an inhibitory motif N1N2GN3G in which at least any two of N1, N2, and N3 are G (guanosine). Dr. Ashman's laboratory described a group of inhibitory 15-mer ODN, having three or four consecutive Gs, that blocked apoptosis protection and cell-cycle entry induced by stimulatory ODN (Lenert et al., 2001; Stunz et al., 2002; Lenert et al., 2003). The immunoinhibitory effect of these ODN was reported to be specific for CpG-ODN and to involve a mechanism other than simple competition for cellular uptake (Stunz et al., 2002). Independently, Klinman and colleagues reported a single immunoinhibitory ODN (Zeuner et al., 2002; Yamada et al., 2002).